Evaluation of Variation Within the Barcode Region of Cytochrome C Oxidase I (COI) for the Detection of Commercial Callinectes Sa
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Evaluation of variation within the barcode region of Cytochrome c Oxidase I (COI) for the detection of commercial Callinectes sapidus Rathbun, 1896 (blue crab) products of non-US origin Amanda M. Windsor1, M. Katherine Moore2, Kimberly A. Warner3, Sarah R. Stadig1 and Jonathan R. Deeds1 1 Office of Regulatory Science, United States Food and Drug Administration, College Park, MD, USA 2 Conservation Biology Division, Forensic Laboratory, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Charleston, SC, USA 3 Oceana, Washington, DC, USA ABSTRACT Callinectes sapidus Rathbun, 1896 is a western Atlantic species with a disjointed natural geographic range from Massachusetts, USA to Venezuela (distribution area 1) and from Alagoas, Brazil to northern Argentina (distribution area 2). It is the only species of portunid crab commercially harvested in the continental United States but is also imported into the US from several Latin American countries, Venezuela and Mexico in particular. In the United States, crab products labeled as “blue crab” and “Product of the USA” may not legally contain other species of crab or C. sapidus not harvested in the United States. The present study documents nucleotide variation within the barcode region of cytochrome c oxidase I (COI) in 417 reference specimens of C. sapidus collected from throughout its natural range. The goal of this study is to determine if this variation can be utilized to detect mislabeled C. sapidus products sold in interstate commerce by comparing genetic signatures in reference Submitted 16 May 2019 specimens to those observed in commercial crabmeat labeled as “Product of the 3 September 2019 Accepted USA” and “Product of Venezuela.” In reference specimens, we observed high levels of Published 5 November 2019 genetic variation in the barcode region. However, three lineages were consistently Corresponding author fi Amanda M. Windsor, observed with signi cant pairwise Fst values between the lineages. Lineage 1 was [email protected] observed throughout the natural geographic range but predominated in the Academic editor continental US and was the only lineage observed in the major crabmeat-producing Rogerio Sotelo-Mundo states (MD, LA, VA, NC). Lineage 2 primarily occurred in the Caribbean region of Additional Information and distribution area 1 but was also infrequently encountered in the South Atlantic Bight Declarations can be found on region of the US coast. Finally, Lineage 3 was only observed in Brazilian waters and page 20 had the lowest haplotype and nucleotide diversity values. Lineages 1 and 2 were DOI 10.7717/peerj.7827 separated by a mean pairwise distance (p-distance) of 3.15%, whereas Lineage 3 had a mean p-distance of 2.55% and 1.35% to Lineages 1 and 2, respectively. Within lineage Distributed under mean p-distances were 0.45%, 0.19%, and 0.07% for Lineages 1, 2, and 3, respectively. Creative Commons CC-BY 4.0 Among all vouchered reference specimens collected from the continental United States, Mexico, Puerto Rico, and Venezuela, we identified 22 phylogenetically How to cite this article Windsor AM, Moore MK, Warner KA, Stadig SR, Deeds JR. 2019. Evaluation of variation within the barcode region of Cytochrome c Oxidase I (COI) for the detection of commercial Callinectes sapidus Rathbun, 1896 (blue crab) products of non-US origin. PeerJ 7:e7827 DOI 10.7717/peerj.7827 informative sites that drive observed lineage divergences. Haplotypes identified from barcode COI sequences from commercial C. sapidus products labeled as originating from the US all aligned with haplotypes from Lineage 1 reference specimens and haplotypes from commercial products labeled as originating from Venezuela all aligned with Lineage 2, suggesting that these lineages may be useful for indicating whether products originate from the continental US or are imported when package labeling is in question. Subjects Aquaculture, Fisheries and Fish Science, Biogeography, Food Science and Technology, Marine Biology, Molecular Biology Keywords Barcoding, Crustacea, Decapoda, Portunidae, Seafood, Species substitution, Swimming crab INTRODUCTION Callinectes sapidus: geographic range, taxonomic history, and commercial importance The natural range of Callinectes sapidus Rathbun, 1896 is the broadest of any species in the genus. This range is disjointed, with one distribution (distribution area 1) extending from Massachusetts, USA south to Venezuela, and a second distribution (distribution area 2) beginning in Alagoas, Brazil and extending south to the northern coast of Argentina (Dos Santos & D’Inaco, 2004; Rodrigues et al., 2017)(Fig. 1). In the Mid-Atlantic region of the US, Maryland and Virginia in particular, C. sapidus has reached iconic status for both its economic and cultural value. Recent northward expansion of the North American range into the Gulf of Maine has been observed and attributed to warmer than usual ocean temperatures in higher latitudes (Johnson, 2015). In addition to its broad natural range, C. sapidus has been introduced to the eastern Atlantic Ocean, Mediterranean, Adriatic, North, and Baltic Seas as well as waters of Hawai’i and Japan via human-mediated transport (Benabdi, Belmahi & Grimes, 2019; Castejón & Guerao, 2013; Dulčić et al., 2011; Eldredge, 1995; Galil, 2008; Keskin & Atar, 2013; Nehring, 2011; Perdikaris et al., 2015; Piras, Esposito & Meloni, 2019; Snovsky & Galil, 1990; Vasconcelos et al., 2019). A review of the taxonomic history of C. sapidus revealed that the species was once divided into two subspecies, the “typical” C. sapidus sapidus and “acute” C. sapidus acutidens Rathbun, 1896 which was defined by its stronger carapace features and larger, more acute spines. This subspecies was first described from small specimens from Brazil and reportedly ranged from the east coast of Florida to the central coast of Brazil (Rathbun, 1930). However, Williams (1974) synomymized the two subspecies after examining over 1,500 specimens. He concluded that external morphology in C. sapidus is highly variable throughout its range and that morphological characters do not reliably segregate individuals putatively identified as C. sapidus acutidens from those identified as “typical” C. sapidus. Williams (1984) went on to characterize C. sapidus as a polymorphic species in which environmental factors may influence external carapace morphology. Harvest of C. sapidus, both whole, live crabs for local consumption and processed crabmeat for domestic distribution, occurs in every US state on the Atlantic and Gulf Windsor et al. (2019), PeerJ, DOI 10.7717/peerj.7827 2/26 Figure 1 Map of natural distribution of C. sapidus along with collection locations and number of specimens successfully sequenced for this study showing proportion of each lineage in each location. Map of natural distribution of C. sapidus along with collection locations and number of spe- cimens successfully sequenced for this study showing proportion of each lineage in each location. Green, Distribution 1; dark blue, Distribution 2; light blue, Lineage 1; pink, Lineage 2; orange, lineage 3; NJ, New Jersey; DE, Delaware; MD, Maryland; VA, Virginia; NC, North Carolina; SC, South Carolina; GA, Georgia; LRFL, Loxahatchee River, Florida; MFL, Miami, Florida; NFL, Naples, Florida; SPFL, St. Petersburg, Florida; PCFL, Panama City, Florida; LA, Louisiana; NTX; northern Texas; STX, southern Texas; VE, Veracruz, México, CM, Campeche, México; YU, Yucatán, Mexico; CR, Costa Rica; ZU, Zulia, Venezuela; FA, Falcón, Venezuela; PR, Puerto Rico; SP, São Paulo, Brazil; RGS, Rio Grande do Sul, Brazil; SCat; Santa Catarina, Brazil. Full-size DOI: 10.7717/peerj.7827/fig-1 coasts from Connecticut to Texas. However, >80% of harvest for production of processed crabmeat occurs in the states of Louisiana, Maryland, Virginia, and North Carolina (NOAA National Marine Fisheries Service Office of Science and Technology CFS, 2017b) (Fig. 2). In addition to domestic US harvest, C. sapidus is imported to the US, primarily as processed crabmeat, from throughout its natural range. Additionally, C. sapidus has become so well established in the eastern Mediterranean Sea that it is being harvested for both local consumption and export (Kevrekidis & Antoniadou, 2018; Piras, Esposito & Meloni, 2019). Crabmeat, regardless of species, is typically processed in such a way that species identification based on visual inspection is impossible, thus providing an opportunity for intentional species substitution and fraud if imported crabmeat is falsely sold to US consumers as domestic C. sapidus at a premium price. Public and political interest in seafood-related fraud in the United States led to the creation in 2014 of the Presidential Task Force on Combating Illegal, Unreported, and Unregulated (IUU) Fishing and Seafood Fraud. As part of its action plan, the taskforce Windsor et al. (2019), PeerJ, DOI 10.7717/peerj.7827 3/26 Figure 2 Landings of C. sapidus in the United States. Proportion of five-year average of US domestic landing of Callinectes sapidus across 15 states. Percent contribution in parentheses. Full-size DOI: 10.7717/peerj.7827/fig-2 published a list of commercially important fishes, crustaceans, and molluscs at risk for IUU fishing and fraud in the Federal Register in 2015 (Presidential Task Force on Combating Illegal Unreported and Unregulated (IUU) Fishing and Seafood Fraud Action Plan, 2014) (80 FR 66867). Of the 13 species or species groups chosen for the initial “at risk” listing, three